Adeno-associated virus mediated B7.1 vaccination synergizes with angiostatin to eradicate disseminated liver metastatic cancers

Abstract

The present invention provides adeno-associated viral (AAV) vectors encoding an angiostatin protein (“AAV-angiostatin vector”) and/or a costimulatory molecule B7.1 (“AAV-B7.1 vector”). The AAV-angiostatin vector can be administered to a subject, alone or in combination, sequentially or simultaneously, with a AAV-B7.1 vector for treatment, management or prevention of metastatic tumors. Pharmaceutical compositions and vaccines comprising the AAV-angiostatin vector and/or the AAV-B7.1 vector and methods of manufacturing are also described. Administration of AAV-angiostatin and AAV-B7.1 vectors by intraportal and muscular injections are also provided.

What is claimed:  
     
         1 . A nucleic acid molecule comprising an adeno-associated viral vector, and a CAG promoter which is operably linked to a nucleic acid sequence encoding angiostatin, wherein the CAG promoter comprises a cytomegalovirus enhancer and beta-actin promoter.  
     
     
         2 . The nucleic acid molecule of  claim 1  further comprising a woodchuck hepatitis B virus post-transcriptional regulatory element.  
     
     
         3 . A nucleic acid molecule comprising an adeno-associated viral vector and a CAG promoter which is operably linked to: (a) the nucleotide sequence of SEQ ID NO:1; or (b) a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:2, wherein the CAG promoter comprises a cytomegalovirus enhancer and beta-actin promoter.  
     
     
         4 . The nucleic acid molecule of  claim 3  further comprising a woodchuck hepatitis B virus post-transcriptional regulatory element.  
     
     
         5 . A vector comprising the nucleic acid molecule of  claim 1 .  
     
     
         6 . A host cell comprising the vector of  claim 5 .  
     
     
         7 . A pharmaceutical composition comprising the nucleic acid molecule of  claim 1 , and a pharmaceutically acceptable carrier.  
     
     
         8 . A nucleic acid molecule comprising an adeno-associated viral vector, and a CAG promoter which is operably linked to a nucleic acid sequence encoding B7.1, wherein the CAG promoter comprises a cytomegalovirus enhancer and beta-actin promoter.  
     
     
         9 . The nucleic acid molecule of  claim 8  further comprising a woodchuck hepatitis B virus post-transcriptional regulatory element.  
     
     
         10 . A nucleic acid molecule comprising an adeno-associated viral vector and a CAG promoter which is operably linked to: (a) the nucleotide sequence of SEQ ID NO:3; or (b) a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:4, wherein the CAG promoter comprises a cytomegalovirus enhancer and beta-actin promoter.  
     
     
         11 . The nucleic acid molecule of  claim 10  further comprising a woodchuck hepatitis B virus post-transcriptional regulatory element.  
     
     
         12 . A vector comprising the nucleic acid molecule of  claim 8 .  
     
     
         13 . A host cell comprising the vector of  claim 12 .  
     
     
         14 . A pharmaceutical composition comprising the nucleic acid molecule of  claim 8 , and a pharmaceutically acceptable carrier.  
     
     
         15 . A method for the production of isolated or purified angiostatin protein, or a fragment, variant, or derivative thereof, said method comprising (i) growing the cell of  claim 6  such that angiostatin protein is expressed; and (ii) isolating or purifying said angiostatin protein.  
     
     
         16 . A method for the production of isolated or purified B7.1 protein, or a fragment, variant, or derivative thereof, said method comprising (i) growing the cell of  claim 13  such that B7.1 protein is expressed; and (ii) isolating or purifying said B7.1 protein.  
     
     
         17 . A method of treating or preventing cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a nucleic acid molecule comprising an adeno-associated viral vector and a CAG promoter which is operably linked to a nucleic acid sequence encoding angiostatin, wherein the CAG promoter comprises a cytomegalovirus enhancer and beta-actin promoter.  
     
     
         18 . A method of treating or preventing cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a nucleic acid molecule comprising an adeno-associated viral vector and a CAG promoter which is operably linked to a nucleic acid sequence encoding B7.1, wherein the CAG promoter comprises a cytomegalovirus enhancer and beta-actin promoter.  
     
     
         19 . A method of treating or preventing cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a nucleic acid molecule comprising an adeno-associated viral vector and a CAG promoter which is operably linked to: (a) the nucleotide sequence of SEQ ID NO:1; or (b) a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:2, wherein the CAG promoter comprises a cytomegalovirus enhancer and beta-actin promoter.  
     
     
         20 . A method of treating or preventing cancer in a subject in need thereof, said method comprising administering a prophylactically effective amount of a nucleic acid molecule comprising an adeno-associated viral vector and a CAG promoter which is operably linked to: (a) the nucleotide sequence of SEQ ID NO:3; or (b) a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:4, wherein the CAG promoter comprises a cytomegalovirus enhancer and beta-actin promoter.  
     
     
         21 . The method of  claim 17  or  18 , wherein the nucleic acid molecule further comprises a woodchuck hepatitis B virus post-transcriptional regulatory element.  
     
     
         22 . The method of  claim 17  or  18 , wherein said cancer is liver cancer.  
     
     
         23 . The method of  claim 22 , wherein said liver cancer is metastatic.  
     
     
         24 . The method of  claim 17  or  18 , wherein the nucleic acid molecule is administered via a portal vein.  
     
     
         25 . The method of  claim 17  or  18 , wherein the nucleic acid molecule is administered by muscular injection.  
     
     
         26 . A method of treating or preventing cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of: 
 (a) a first nucleic acid molecule comprising an adeno-associated viral vector and a CAG promoter which is operably linked to a nucleic acid sequence encoding angiostatin, wherein the CAG promoter comprises a cytomegalovirus enhancer and beta-actin promoter; and    (b) a second nucleic acid molecule comprising an adeno-associated viral vector and a CAG promoter which is operably linked to a nucleic acid sequence encoding B7.1, wherein the CAG promoter comprises a cytomegalovirus enhancer and beta-actin promoter.    
     
     
         27 . A method of treating or preventing cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of: 
 (a) a first nucleic acid molecule comprising an adeno-associated viral vector and a CAG promoter which is operably linked to: (a) the nucleotide sequence of SEQ ID NO:1; or (b) a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:2, wherein the CAG promoter comprises a cytomegalovirus enhancer and beta-actin promoter; and    (b) a second nucleic acid molecule comprising an adeno-associated viral vector and a CAG promoter which is operably linked to: (a) the nucleotide sequence of SEQ ID NO:3; or (b) a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:4, wherein the CAG promoter comprises a cytomegalovirus enhancer and beta-actin promoter.    
     
     
         28 . The method of  claim 26 , wherein the first nucleic acid molecule further comprises a woodchuck hepatitis B virus post-transcriptional regulatory element.  
     
     
         29 . The method of  claim 26 , wherein the second nucleic acid molecule further comprises a woodchuck hepatitis B virus post-transcriptional regulatory element.  
     
     
         30 . The method of  claim 26 , wherein said cancer is liver cancer.  
     
     
         31 . The method of  claim 30 , wherein said liver cancer is metastatic.  
     
     
         32 . The method of  claim 26 , wherein the first nucleic acid molecule and second nucleic acid molecule are administered via a portal vein.  
     
     
         33 . The method of  claim 26 , wherein the first nucleic acid molecule and second nucleic acid molecule are administered by muscular injection.  
     
     
         34 . The method of  claim 26 , wherein the first nucleic acid molecule and the second nucleic acid molecule are administered sequentially.  
     
     
         35 . The method of  claim 26 , wherein the first nucleic acid molecule and the second nucleic acid molecule are administered simultaneously.  
     
     
         36 . A nucleic acid molecule comprising an adeno-associated viral vector, and a CAG promoter which is operably linked to a first polynucleotide comprising a first nucleic acid sequence encoding angiostatin, and a second polynucleotide comprising a second nucleic acid sequence encoding B7.1, wherein the CAG promoter comprises a cytomegalovirus enhancer and beta-actin promoter.  
     
     
         37 . The nucleic acid molecule of  claim 36  further comprising a woodchuck hepatitis B virus post-transcriptional regulatory element.  
     
     
         38 . A nucleic acid molecule comprising an adeno-associated viral vector and a CAG promoter which is operably linked to: (a) a first polynucleotide comprising (i) the nucleotide sequence of SEQ ID NO:1, or (ii) a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:2; and (b) a second polynucleotide comprising (i) the nucleotide sequence of SEQ ID NO:3, or (ii) a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:4, wherein the CAG promoter comprises a cytomegalovirus enhancer and beta-actin promoter  
     
     
         39 . The nucleic acid molecule of  claim 38  further comprising a woodchuck hepatitis B virus post-transcriptional regulatory element.  
     
     
         40 . A vector comprising the nucleic acid molecule of  claim 36 .  
     
     
         41 . A host cell comprising the vector of  claim 40 .  
     
     
         42 . A pharmaceutical composition comprising the nucleic acid molecule of  claim 36 , and a pharmaceutically acceptable carrier.  
     
     
         43 . A method for the production of isolated or purified B7.1 protein and angiostatin, or a fragment, variant, or derivative thereof, said method comprising (i) growing the cell of  claim 41  such that B7.1 protein and angiostatin are expressed; and (ii) isolating or purifying said B7.1 protein and angiostatin.  
     
     
         44 . A method of treating or preventing cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a nucleic acid molecule comprising an adeno-associated viral vector, and a CAG promoter which is operably linked to a first polynucleotide comprising a first nucleic acid sequence encoding angiostatin, and a second polynucleotide comprising a second nucleic acid sequence encoding B7.1, wherein the CAG promoter comprises a cytomegalovirus enhancer and beta-actin promoter.  
     
     
         45 . A method of treating or preventing cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a nucleic acid molecule comprising an adeno-associated viral vector and a CAG promoter which is operably linked to: (a) a first polynucleotide comprising (i) the nucleotide sequence of SEQ ID NO:1, or (ii) a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:2; and (b) a second polynucleotide comprising (i) the nucleotide sequence of SEQ ID NO:3, or (ii) a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:4, wherein the CAG promoter comprises a cytomegalovirus enhancer and beta-actin promoter.  
     
     
         46 . The method of  claim 44 , wherein the first polynucleotide further comprises a woodchuck hepatitis B virus post-transcriptional regulatory element.  
     
     
         47 . The method of  claim 44 , wherein the second polynucleotide further comprises a woodchuck hepatitis B virus post-transcriptional regulatory element.  
     
     
         48 . The method of  claim 44 , wherein said cancer is liver cancer.  
     
     
         49 . The method of  claim 48 , wherein said liver cancer is metastatic.  
     
     
         50 . The method of  claim 44 , wherein the nucleic acid molecule is administered via a portal vein.  
     
     
         51 . The method of  claim 44 , wherein the nucleic acid molecule is administered by muscular injection.